[Frontiers in Bioscience E3, 1337-1348, June 1, 2011]
Acylated catechin derivatives: inhibitors of DNA polymerase and angiogenesis
Yoshiyuki Mizushina1,2, Akiko Saito3, Keiko Horikawa4, Noriyuki Nakajima4, Akira Tanaka5, Hiromi Yoshida1,2, Kiminori Matsubara2,6
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TABLE OF CONTENTS
1. ABSTRACT
Catechins in green tea display anti-cancer and anti-angiogenesis activities. We previously found that some catechins, such as epigallocatechin-3-O-gallate (EGCG), inhibit the activities of eukaryotic DNA polymerases (pols) (Y. Mizushina et al.: Structural analysis of catechin derivatives as mammalian DNA polymerase inhibitors. Biochem Biophys Res Commun 333, 101-109 (2005)). In this study, we discuss the effects of chemical modifications of catechin and epicatechin that enhance their anti-cancer and anti-angiogenic activities based on pol inhibition. Catechins conjugated with fatty acid (3-O-acylcatechins) are stronger inhibitors of mammalian pol than epicatechins conjugated with fatty acid (3-O-acylepicatechins). Moreover, 3-O-acylcatechins are more potent inhibitors of cultured cell growth both of the human colon carcinoma cell line (HCT116 cells) and human umbilical vein endothelial cell (HUVEC) line, as well as angiogenesis by comparison with 3-O-acylepicatechins. Catechin conjugated with stearic acid ((2R,3S)-3',4',5,7-tetrahydroxyflavan-3-yl octadecanoate; C-C18) was the strongest inhibitor in replicative pol alpha and repair-related pol beta, as well as the cultured cell growth and angiogenesis assays in the compounds tested. C-C18 also suppressed HUVEC tube formation on reconstituted basement membrane suggesting that it affected not only pols but also signal transduction pathways in HUVECs. These data indicate that the acylated catechins target both pols and angiogenesis as anti-cancer agents. Moreover, the results suggest that acylation of catechin is an effective chemical modification to improve the anti-cancer activity of catechin.
2. INTRODUCTION
Natural polyphenols distributed in plants have diverse biological activities: anti-oxidant, anti-cancer, and anti-inflammation activities (1). Recent evidence shows that polyphenols have pharmacological effects and are useful models for the design of new drugs. Green tea catechins are a particularly attractive set of polyphenols because they have been shown to lower the risk of various cancers and suppress cancer cell growth and angiogenesis (2). Angiogenesis is involved in tumor growth and metastasis, atherosclerosis and diabetic retinopathy (3). Recently, significant resources have been directed towards developing more effective angiogenesis inhibitors (4). We have identified some angiogenesis inhibitors in natural products including vitamin B6 (5-7), algal polysaccharides (8, 9), algal carotenoids (10, 11) and phenolic compounds (12). Recently, we have reported that some DNA polymerase (pol) inhibitors, such as a polyphenolic compound (13), glycolipids from spinach (14) as well as vitamin K3 (15) and its derivatives (16, 17), have a strong inhibitory effect on angiogenesis in vitro.
Among DNA metabolic enzymes, pol catalyzes the addition of deoxyribonucleotides to the 3'-hydroxyl terminus of primed double-stranded DNA (dsDNA) molecules (18). The human genome encodes 15 pols that conduct cellular DNA synthesis (19). Eukaryotic cells reportedly contain three replicative types, namely pols alpha, delta and epsilon; mitochondrial pol gamma; and thirteen repair types, namely pols beta, delta, epsilon, zeta, eta, theta, iota, kappa, lambda, mu and nu, REV1 and terminal deoxynucleotidyl transferase (TdT) (20). DNA metabolic enzymes such as pols are not only essential for DNA replication, repair and recombination, but are also involved in cell division. Selective inhibitors of eukaryotic pols are considered as a group of potentially useful anti-cancer and anti-parasitic agents, because some inhibitors suppress human cancer cell proliferation and have cytotoxicity (21-23).
We previously reported that certain catechins, such as epigallocatechin-3-O-gallate (EGCG), inhibit the activities of mammalian pols (24). Recently, our efforts have been directed towards the synthesis of potent catechin and epicatechin derivatives conjugated with fatty acids, such as 3-O-acylcatechins and 3-O-acylepicatechins, respectively. In this study, the characteristics of the acylated catechin and epicatechin derivatives as pol and angiogenesis inhibitors are discussed.
3. MATERIALS AND METHODS
3.1. Materials
(+)-Catechin (C) and (-)-epicatechin (EC) were produced by Mitsui Norin Co. Ltd. (Shizuoka, Japan). The acylated catechin and epicatechin derivatives (3-O-acyl-flavan-3-ols), which are conjugated fatty acids, were chemically synthesized as described previously (25, 26). The structures of all 15 synthesized compounds are shown in Figure 1. Nucleotides, such as (3H)-deoxythymidine 5'-triphosphate (dTTP) (43 Ci/mmol), and chemically synthesized DNA template, such as poly(dA), were purchased from GE Healthcare Bio-Sciences (Little Chalfont, UK). DNA primer, such as oligo(dT)18, was customized by Sigma-Aldrich K. K. (Hokkaido, Japan). Human recombinant vascular endothelial growth factor (VEGF) was obtained from R&D systems (MN, USA). Other reagents were special grade as commercially available.
3.2. Preparation of DNA polymerases and other DNA metabolic enzymes
Pol alpha was purified from calf thymus by immuno-affinity column chromatography, as described by Tamai et al. (27). Recombinant rat pol beta was purified from Escherichia coli JMpB5, as described by Date et al. (28). The human pol gamma catalytic gene was cloned into pFastBac. Histidine-tagged enzyme was expressed using the BAC-TO-BAC HT Baculovirus Expression System according to the supplier's manual (Life Technologies, MD, USA) and purified using ProBoundresin (Invitrogen Japan, Tokyo, Japan) (29). Human pols delta and epsilon were purified by nuclear fractionation of human peripheral blood cancer cells (Molt-4) using the second subunit of pols delta and epsilon-conjugated affinity column chromatography, respectively (30). Fruit fly pols alpha, delta and epsilon were purified from early embryos of Drosophila melanogaster, as described by Aoyagi et al. (31, 32). Budding yeast (Saccharomyces cerevisiae) pol alpha was purified by ion-exchange column chromatography as described by Hamatake et al. (33). Pol alpha from a higher plant, cauliflower inflorescence, was purified according to the methods outlined by Sakaguchi et al. (34). The Klenow fragment of pol I from E. coli were purchased from Worthington Biochemical Corp. (Freehold, NJ, USA). Taq pol, T4 pol, T7 RNA polymerase and T4 polynucleotide kinase were purchased from Takara Bio (Tokyo, Japan). Bovine pancreas DNase I were obtained from Stratagene Cloning Systems (La Jolla, CA, USA).
3.3. DNA polymerase assays
The reaction mixtures for pol alpha, pol beta, plant pols and prokaryotic pols were described previously (35-37); those for pol gamma, and pols delta and epsilon were as described by Umeda et al. (29) and Ogawa et al. (38), respectively. For pols, poly(dA)/oligo(dT)18 (A/T = 2/1) and 2'-deoxythymidine 5'-triphosphate (dTTP) were used as the DNA template-primer and nucleotide (i.e., 2'-deoxynucleoside 5'-triphosphate (dNTP)) substrate, respectively.
The compounds were dissolved in distilled dimethyl sulfoxide (DMSO) at various concentrations and sonicated for 30 sec. Aliquots of 4 uL sonicated samples were mixed with 16 uL of each enzyme (final amount 0.05 units) in 50 mM Tris-HCl (pH7.5) containing 1 mM dithiothreitol, 50% glycerol and 0.1 mM EDTA, and kept at 0˚C for 10 min. These inhibitor-enzyme mixtures (8 uL) were added to 16 uL of each of the enzyme standard reaction mixtures (final concentration of 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 1 mM MgCl2, 15% glycerol, 10 uM poly(dA)/oligo(dT)18 and 10 uM (3H)-dTTP), and incubation was carried out at 37˚C for 60 min, except for Taq pol, which was incubated at 74˚C for 60 min. Activity without the inhibitor was considered 100%, and the remaining activity at each concentration of the inhibitor was determined relative to this value. One unit of pol activity was defined as the amount of enzyme that catalyzed the incorporation of 1 nmol dNTP (i.e., dTTP) into synthetic DNA template-primers in 60 min at 37˚C under the normal reaction conditions for each enzyme (35-37).
3.4. Other DNA metabolic enzymes assays
The activities of calf primase of pol alpha, T7 RNA polymerase, T4 polynucleotide kinase and bovine DNase I were measured in standard assays according to the manufacturer's specifications, as described by Tamiya-Koizumi et al. (39), Nakayama et al. (40), Soltis et al. (41) and Lu et al. (42), respectively.
3.5. Cell culture and measurement of cell viability
The HCT116 human colon carcinoma cell line and HUVEC line were obtained from American Type Culture Collection (ATCC) (Manassas, VA). HCT116 cells were cultured in McCoy's 5A Medium supplemented with 10% foetal bovine serum, penicillin (100 units/mL) and streptomycin (100 mg/mL). HUVECs were cultured in Eagle's Minimum Essential Medium (MEM) supplemented with 4.5 g of glucose per liter plus 10% fetal calf serum, 5 mM L-glutamine, 50 units/mL penicillin and 50 units/mL streptomycin. These cells were cultured at 37˚C in a humid atmosphere of 5% CO2 / 95% air. For the cell growth assay, the cells were plated at 1 x 104 cells into each well of 96-well microplates with various concentrations of acyl-catechin compound. These compounds were dissolved in DMSO at a concentration of 10 mM as a stock solution. The stock solutions were diluted to the appropriate final concentrations with growth medium as 0.5 % DMSO just before use. The cell viability was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay (43).
3.6. Ex vivo angiogenesis assay
Male Wistar rats (6 week old, Clea Japan, Inc., Tokyo, Japan) were housed two to a metal cages in a room with controlled temperature (24 � 1˚C) and a 12-h light : dark cycle (lights on, 08:00-20:00 h). They had free access to diets and deionized water. The rats were maintained according to the "Guide for the Care and Use of Laboratory Animals" established by Hiroshima University. The ex vivo angiogenesis assay was performed according to slightly modified methods as described before (44, 45). Briefly, a male Wistar rat (body weight ~ 200 g) was sacrificed by bleeding from the right femoral artery under anesthesia with diethyl ether. A thoracic aorta was removed and washed with RPMI 1640 medium to avoid contamination with blood. It was then turned inside out, and cut into short segments about 1-1.5 mm. Collagen gel (gel matrix solution) was made with 8 volumes of porcine tendon collagen solution (3 mg/ml) (Cellmatrix Ia, Nitta Gelatin Co., Osaka, Japan), 1 volume of 10�Eagle's MEM (Gibco, New York, USA), 1 volume of reconstitution buffer (0.08 M NaOH and 200 mM HEPES). These solutions were mixed gently at 4˚C. Each aortic segment was placed in the center of a well on a 6-well culture plate and covered with 0.5 mL of gel matrix solution reconstituted as described. The solution was allowed to gel at 37˚C for 20 min. Culture medium (RPMI 1640 medium (Gibco, New York, USA) containing 1% of ITS+ (Becton Dickinson Labware, MA, USA)) with various concentration of acyl-catechin or vehicle (DMSO) was prepared as the same way described above. Then, the collagen gel was overlaid with 2 mL of culture medium. Incubation was carried out for 10 days in a fully humidified system of 5 % CO2 in the air at 37˚C. The medium was changed on day 7 of the culture. An estimation of the length of the capillary was performed under phase-contrast microscopy by measuring the distance from the cut end of the aortic segment to the approximate mean point of capillary. Microscopic fields were photographed with a digital camera (Nikon, COOLPICKS 950). The length of the capillary was measured using Adobe Photoshop software. Each reported value represents the average of three culture samples.
3.7. Endothelial cells
HUVECs were purchased from Kurabo Industries (Osaka, Japan). Cells were grown in the medium, HuMedia EG 2 (Kurabo Industries, Osaka, Japan), which was modified MCDB 131 medium containing 2% fetal bovine serum (FBS), 10 ng/ml recombinant human epidermal growth factor (EGF), 1 ug/mL hydrocortisone, 50 ug/mL gentamicin, 50 ng/mL amphotericin B, 5 ng/mL recombinant human basic fibroblast growth factor (bFGF) and 10 ug/mL heparin, at 37˚C in a humidified 5% CO2. Subcultures were obtained by treating the HUVEC cultures with 0.025% trypsin-0.01% EDTA solution. HUVEC at passage three to five were used in this experiment.
3.8. HUVEC tube formation assay
Tube formation assay was performed using an In vitro Angiogenesis Assay Kit (Chemicon International, Inc., Temecula, CA, U.S.A.). Briefly, solid gels were prepared according to the manufacture manual on a 96-well tissue culture plate. HUVECs (1 X 105 cells/mL) in HuMedia EG-2 medium containing acyl-catechin (1-100 uM) or vehicle (DMSO) were seeded 100 uL per well onto the surface of the solid gel, ECMatrixTM. The cells were incubated for 12 h at 37˚C in a CO2 incubator. Tube formation was observed under an inverted light microscope at 40 X magnification. Microscopic fields were photographed with a digital camera (Nikon, COOLPICKS 950). The total length of tube structures in each photograph was measured using Adobe Photoshop software. Each reported value represents the average of three samples.
4. RESULTS
4.1. In vitro DNA polymerase inhibition
4.1.1. Effect of the derivatives of 3-O-acylcatechin and 3-O-acylepicatechin on the activities of mammalian pol activity
The 15 compounds of the acylated catechin and epicatechin derivatives (3-O-acyl-flavan-3-ols) are prepared (Figure 1). (+)-Catechin (C) and its acylated derivatives are compound 1 and compounds 2-9, respectively, and (-)-epicatechin (EC) and its acylated derivatives are compound 10 and compounds 11-15, respectively. The purification grade of each compound was >98%. The inhibitory activity of calf pol alpha and rat pol beta against each compound was then investigated. For mammalian pols, pols alpha and beta were used as representative replicative pol and repair-related pols, respectively (19, 20).
In the presence of 25 uM of catechin (compound 1) or epicatechin (compound 10) there was no detectable effect on the activities of pols alpha and beta, whereas the same concentration of acylated derivatives caused significant inhibition of enzyme activity (Figure 2). The inhibitory effect of the derivatives conjugated with longer chain fatty acids was stronger than that of the corresponding shorter acylated derivatives. Compound 8 ((2R,3S)-3',4',5,7-tetrahydroxyflavan-3-yl octadecanoate; C-C18), which is conjugated with C18-fatty acid (stearic acid), was the most potent inhibitor tested. Interestingly, compound 5 (C-C12), which is the C12-fatty acid acylated catechin derivative, inhibited the activities of pols alpha and beta, whereas compound 9 (C-C12Ac), which is the acetylated compound 5, had no such inhibitory effect. These results suggested that both the acyl-chain group and hydroxyl group of catechin are essential for the inhibitory activity. For the 3-O-acylcatechins derivatives (compounds 1-9), the effect of the nine compounds can be ranked as follows: compound 8 (C-C18) > compound 7 (C-C16) > compound 6 (C-C14) > compound 5 (C-C12) > compound 4 (C-C10) > compound 3 (C-C8) > compound 2 (C-C6) > compound 1 (C) = compound 9 (C-C12Ac). When the acyl-chain of the catechin derivative was equivalent to that of the epicatechin derivatives, 3-O-acylcatechins (compounds 2-8) were always found to be stronger inhibitors of pols than 3-O-acylepicatechins (compounds 11-14). Therefore, the stereochemistry of the 3-O-position in the catechin may be important for the inhibition. The inhibitory effect of these compounds on pol alpha showed almost the same tendency as that observed for pol beta. When activated DNA (i.e., DNA digested by bovine deoxyribonuclease I) and dNTP were used as the DNA template-primer and nucleotide substrate, respectively, instead of poly(dA)/oligo(dT)18 (A/T = 2/1) and dTTP, the mode of inhibition by these compounds did not change (data not shown).
4.1.2. Effect of compound 8 (C-C18) on various pols and other DNA metabolic enzymes
Of all the compounds (compounds 1-15) investigated in this study compound 8 (C-C18) was the strongest inhibitor of mammalian pols alpha and beta (Figure 2). Therefore, we decided to focus our studies on compound 8. The IC50 values for the inhibition of DNA replicative pols, such as pols alpha, delta and epsilon, mitochondrial pol gamma, and DNA repair-related pol beta for compound 8 ranged between 9.4-14.1 uM (Table 1). The pol alpha inhibitory effect of this compound was the strongest among these five major classes of mammalian pols tested. Compound 8 also inhibited the activities of pols alpha, delta and epsilon from insect (Drosophila melanogaster) and pol alpha from protistan (Saccharomyces cerevisiae) as well as mammalian pols. By contrast, this compound did not inhibit the activity of pol alpha from plant (cauliflower), prokaryotic pols, such as Escherichia coli pol I (Klenow fragment), Taq pol and T4 pol, and other DNA-metabolic enzymes such as calf primase of pol alpha, T7 RNA polymerase, T4 polynucleotide kinase and bovine deoxyribonuclease I (DNase I). As a result, compound 8 was shown to be a potent and selective inhibitor of mammalian pols, especially DNA replicative pol alpha.
To determine whether the inhibition resulted from binding to DNA or binding to the enzyme, the interaction of compound 8 with dsDNA was investigated by measuring the thermal transition of dsDNA with or without the compound. The melting temperature (Tm) of dsDNA mixed with an excess amount of compound 8 (200 uM) was measured using a spectrophotometer equipped with a thermoelectric cell holder. In the temperature range (75-98˚C) used, no thermal transition (Tm) was observed. By contrast, 15 uM of ethidium bromide (EtBr), a typical intercalating compound that was used as a positive control, produced a clear thermal transition. These observations indicate that compound 8 binds directly to the enzyme to inhibit its activity, rather than intercalating with the DNA as a template-primer.
4.1.3. Mode of inhibition of mammalian pols alpha and beta by compound 8 (C-C18)
Next, to elucidate the mechanism by which compound 8 (C-C18) inhibits mammalian pols alpha and beta, the extent of inhibition as a function of the DNA template-primer or dNTP substrate concentrations was studied. In kinetic analysis, poly (dA)/oligo(dT)18 and dTTP were used as the synthetic DNA template-primer and dNTP substrate, respectively. Double reciprocal plots of the obtained data show that compound 8-induces inhibition of the activities of both pol alpha and pol beta in a competitive and non-competitive fashion with the DNA template-primer and the dNTP substrate, respectively (Table 2). These results suggest that the compound binds directly to the DNA template binding site of the enzyme. The inhibition constant (Ki) values of compound 8, obtained from Dixon plots, were found to be 3.6-7.4 uM and 5.3-9.3 uM for the DNA template-primer and dTTP, substrate, respectively. Because the Ki values for the DNA template-primer were smaller than those for the dNTP substrate, the affinity of compound 8 must be greater for the enzyme-DNA template-primer binary complex than for the enzyme-nucleotide substrate complex.
4.2. Suppression of cultured cell growth
Pols have recently emerged as important cellular targets for chemical intervention in the development of anti-cancer agents. Therefore, these synthesized catechin derivatives (compounds 1-15) are promising chemotherapeutic candidates. To clarify the cytological effects of these compounds, their influence on survival of two cultured cell lines, such as human colon carcinoma (HCT116) cells and human umbilical vein endothelial cells (HUVECs), was tested by MTT assay (43). Anti-cancer chemotherapy agents inhibit the growth of human cancer cells such as HCT116 cells, and many inhibitors of angiogenesis suppress the proliferation of endothelial cells such as HUVECs. As shown in Figure 3, neither 25 uM of compound 1 (C), compound 10 (EC) and their acetylated derivatives (compounds 9 and 15) influenced the growth of HCT116 cells. However, 25 uM of the derivatives of 3-O-acylcatechins (compounds 2-8) and 3-O-acylepicatechins (compounds 11-14) suppressed cell growth. Compound 8 (C-C18) had the strongest growth inhibitory effect on the HCT116 cancer cell line among the compounds tested, and the LD50 value was 12.5 uM. This compound also dose-dependently suppressed the growth of other human cancer cell lines such as A549 (human lung cancer), BALL-1 (human leukemia), HL-60 (human promyelocytic carcinoma), Molt-4 (human leukemia) and NUGC-3 (human gastric cancer) with a range of LD50 values of between 10-30 uM (data not shown). Intriguingly, the LD50 value of compound 8 is approximately the same as the IC50 value for mammalian pol activity, suggesting that the growth inhibition is primarily a function of the effect of compound 8 on the replicative pols, such as pol alpha. The growth suppression of these compounds on HUVECs showed the same tendency as that on HCT116 cells, but the inhibitory effect on HUVECs was stronger. A significant correlation was found between the cultured cell proliferation and the inhibition of mammalian pols such as pol alpha (see Figure 2 and Figure 3). The acylated catechin and epicatechin derivatives may be able to penetrate the cells more efficiently to reach the nucleus and interact with pol alpha. The resultant inhibition of pol alpha activity by the compound may lead to the observed cell growth suppression. The effect of the various compounds on angiogenesis will be addressed in the next part of this study.
4.3. Angiogenesis inhibition
4.3.1. Effect of the derivatives of 3-O-acylcatechin and 3-O-acylepicatechin on ex vivo angiogenesis
It has been shown that tea catechins have anti-angiogenic activity (46). Among the tea catechins, EGCG is the most effective anti-angiogenic agent. The ex vivo anti-angiogenic effect of compounds 1-15 was investigated in a rat aortic ring model (Figure 4). This angiogenesis method is widely used to evaluate anti-angiogenic agents (47-49). In this model, fibroblastic fusiform cells migrate from the ends of the aortic rings over a period of 2 to 3 days and spread in the collagen gel and then microvessels appear from the ends of aortic rings after 5 to 6 days (Figure 5A). Interestingly, the inhibitory effects of all the acylated derivatives of catechins (compounds 2-8) and epicatechins (compounds 12 and 13) were stronger than catechin (compound 1) and epicatechin (compound 10), respectively (Figure 4). Moreover, the inhibitory action increased with the length of the acyl moiety. 3-O-Acylcatechin derivatives were stronger suppressors of the ex vivo angiogenesis than 3-O-acylepicatechin derivatives. The anti-angiogenic activity by these compounds was correlated with pol inhibition (Figure 2) and suppression of the cultured cell proliferation (Figure 3). Compound 6 (C-C14) and compound 7 (C-C16) completely inhibited microvessel outgrowth, although fibroblastic fusiform cells migrated from the ends of the aortic rings (data not shown). By contrast, compound 8 (C-C18) completely suppressed fibroblastic fusiform cell migration and microvessel outgrowth as shown in Figure 5A. Therefore, compound 8 was the most effective inhibitor of angiogenesis. Moreover, compound 8 inhibited angiogenesis in a dose-dependent manner and was statistically significant at greater than 50 uM (Figure 5B).
4.3.2. Effect of compound 8 (C-C18) on HUVEC tube formation
The effect of compound 8 (C-C18) was examined in an in vitro angiogenesis model using HUVECs. HUVECs inoculated on a reconstituted basement membrane (Matrigel™) migrated, then attached to each other, and finally formed tube structures (Figure 6A). Compound 8 suppressed HUVEC tube formation in a dose-dependent manner (Figure 6A and B). Vascular endothelial growth factor (VEGF) strongly stimulated HUVEC migration, and the effect of compound 8 on HUVEC migration on gelatin coated Boyden chambers was examined. Compound 8 did not affect HUVEC migration induced by VEGF at the examined concentration of 1 to 100 uM (data not shown). Taken together, the above results show that compound 8 exerts its anti-angiogenic activity through inhibiting pol activity and endothelial cell tube formation.
5. DISCUSSION
Many organisms are known to contain as many as 15 types of pol (19), which catalyze DNA replication, repair and recombination (19, 20). Pols, especially pol alpha, are regarded as the target of some anti-cancer drugs because they play a central role in DNA replication, which is indispensable for the proliferation of cancer cells. Thus, pol inhibitors are promising candidates as anti-cancer chemotherapy agents. Based on this idea, we have isolated many new pol inhibitors over the past 15 years from natural sources, particular from food materials (22, 50). Moreover, we have chemically synthesized the pol inhibitors and related compounds.
A series of acylated derivatives of catechin (compounds 1-9) and epicatechin (compounds 10-15), which are polyphenols found in green tea, were used in this study (Figure 1). These compounds were then investigated to determine the relationship between their chemical structure and respective anti-cancer activities. These results indicate that catechin derivatives (compounds 2-8) exert stronger inhibitory effects on pol activity, cultured cell proliferation and angiogenesis compared to epicatechin derivatives (compounds 11-14) (Figures 2-4). Compound 8 (C-C18) was the strongest inhibitor among the synthesized acylated catechin derivatives. The in vitro inhibition of cancer cell proliferation and induction of anti-angiogenesis by compound 8 may be caused by the inhibition of pols, especially replicative pol alpha. We are trying to investigate whether the specificity of compound 8-mediated inhibition of mammalian pol activity is another major concern for further studies.
Angiogenesis is the formation of new blood vessels from pre-existing blood vessels. The process of angiogenesis is involved in various pathological states including solid tumor growth, diabetic retinopathy, and age-related macular degeneration (3). Inhibition of angiogenesis is an important target for cancer treatment because metastasis as well as solid tumor growth is facilitated through angiogenesis (4). Interestingly, administration of drugs exhibiting anti-angiogenic activity has been shown to reduce the risk of brain diseases in older patients (51). Given that angiogenesis-related diseases pose serious medical problems worldwide, there has been much research devoted to developing effective angiogenesis inhibitors.
Various biological activities of natural polyphenols have been reported. It has been shown that catechins in green tea lower the risk of cancer and cardiovascular diseases (2). EGCG is the most abundant compound present in green tea and has been the most extensively studied in terms of its biological action. EGCG has strong anti-cancer and anti-angiogenic effects (52). In addition, it has been shown that esterification of epigallocatechin increases anti-virus activity (53). Moreover, epicatechin and its metabolite protect human fibroblast cells from oxidative-stress-induced cell death (54). These observations suggest that chemical modification of green tea catechins could improve the biological activity of epigallocatechin.
It has been demonstrated that EGCG strongly inhibits endothelial cell tube formation, but catechin (50 uM) exerts no effect (55). The different effects can be ascribed to their respective action on the phosphorylation of the VEGF receptor. EGCG inhibits tyrosine phosphorylation of the VEGF receptor, whereas catechin does not (55). HUVECs migrate, attach to each other, and form tube-like structures on reconstituted basement membrane. This morphological change is an important process in forming new blood vessels. Although compound 8 strongly inhibits HUVEC tube formation (Figures 5 and 6), it does not affect HUVEC migration stimulated by VEGF (data not shown). This result suggests that compound 8 does not inhibit VEGF receptor phosphorylation. Therefore, compound 8 appears to target the interaction of endothelial cells or signal transduction pathways involved in endothelial cell tube formation. Although the exact mechanism by which compound 8 inhibits pol activity is unknown, the present study suggests that the stereochemistry of the fatty acid moiety is an important factor that affects the formation of the DNA template-primer complex or configuration of the catalytic domain (24, 56). The acylated derivatives of catechin and epicatechin exert a growth suppressive effect on the cultured cells, such as HCT116 cancer cells and HUVECs, by inhibiting pol activity, which are critical enzymes for cell proliferation. In addition, compound 8 strongly inhibits HUVEC tube formation indicating that this compound can target other molecules involved in neovascularization.
We synthesized several derivatives of catechin and epicatechin, such as 3-O-acylcatechin and 3-O-acylepicatechin, respectively (3-O-acyl-flavan-3-ols) (25, 26). 3-O-Acylcatechin derivatives have been synthesized previously (53, 57) in order to improve their physical properties, such as the solubility of hydrophilic catechins in lipidic matrices. Among the hydroxy groups of catechin derivatives, modification at the 3'-position of the C-ring is most suitable for this purpose. In addition, the phenolic hydroxy groups, especially on the B-ring, are important for the anti-oxidant activity of catechin derivatives. Indeed, it is reported that the inhibitory activity of the activation of the Epstein-Barr virus early antigen (EBV-EA) is increased by esterification of epigallocatechin with fatty acid (53). These findings led us to surmise whether esterification of catechin with fatty acid might enhance anti-cancer activity. Thus, acylated catechin derivatives with a long alkyl chain, such as compound 8, are promising candidates for developing a novel series of anti-angiogenic agents.
6. ACKNOWLEDGEMENTS
We are grateful for the donation of calf pol alpha by Dr. M. Takemura of Tokyo University of Science (Tokyo, Japan); rat pol beta, human pols delta and epsilon by Dr. K. Sakaguchi of Tokyo University of Science (Chiba, Japan); and human pol gamma by Dr. M. Suzuki of Nagoya University School of Medicine (Nagoya, Japan). This work was supported in part by a Grant-in-aid for Kobe-Gakuin University Joint Research (A), and "Academic Frontier" Project for Private Universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2006-2010, (H. Y. and Y. M.). Y. M. acknowledges a Grant-in-Aid for Young Scientists (A) (No. 19680031) from MEXT.
7. REFERENCES
1. C. S. Yang, J. M. Landau, M. T. Huang and H. L. Newmark: Inhibition of carcinogenesis by dietary polyphenolic compounds. Annu Rev Nutr 21, 381-406 (2001)
doi:10.1146/annurev.nutr.21.1.381
PMid:11375442
2. C. S. Yang, J. Y. Chung, G. Yang, S. K. Chhabra and M. J. Lee: Tea and tea polyphenols in cancer prevention. J Nutr 130, 472S-478S (2000)
PMid:10721932
3. J. Folkman: Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1, 27-31 (1995)
doi:10.1038/nm0195-27
PMid:7584949
4. Y. Cao: Antiangiogenic cancer therapy. Semin Cancer Biol 14, 139-145 (2004)
doi:10.1016/j.semcancer.2003.09.018
PMid:15018898
5. K. Matsubara, M. Mori, Y. Matsuura and N. Kato: Pyridoxal 5'-phosphate and pyridoxal inhibit angiogenesis in serum-free rat aortic ring assay. Int J Mol Med 8, 505-508 (2001)
PMid:11605018
6. S. Komatsu, N. Yanaka, K. Matsubara and N. Kato: Antitumor effect of vitamin B6 and its mechanisms. Biochim Biophys Acta 1647, 127-130 (2003)
PMid:12686121
7. K. Matsubara, S. Komatsu, T. Oka and N. Kato: Vitamin B6-mediated suppression of colon tumorigenesis, cell proliferation, and angiogenesis (review). J Nutr Biochem 14, 246-250 (2003)
doi:10.1016/S0955-2863(03)00009-3
8. K. Matsubara, M. Mori, H. Matsumoto, K. Hori and K. Miyazawa: Antiangiogenic properties of a sulfated galactan isolated from a marine alga, Codium cylindricum. J Appl Phycol 15, 87-90 (2003)
doi:10.1023/A:1022958222915
9. K. Matsubara, C. Xue, X. Zhao, M. Mori, T. Sugawara and T. Hirata: Effects of middle molecular weight fucoidans on in vitro and ex vivo angiogenesis of endothelial cells. Int J Mol Med 15, 695-699 (2005)
PMid:15754034
10. T. Sugawara, K. Matsubara, R. Akagi, M. Mori and T. Hirata: Antiangiogenic activity of brown algae fucoxanthin and its deacetylated product, fucoxanthinol. J Agric Food Chem 54, 9805-9810 (2006)
doi:10.1021/jf062204q
PMid:17177505
11. P. Ganesan, K. Matsubara, T. Ohkubo, Y. Tanaka, K. Noda, T. Sugawara and T. Hirata: Anti-angiogenic effect of siphonaxanthin from green alga, Codium fragile. Phytomedicine, in press.
12. K. Matsubara, T. Kaneyuki, T. Miyake and M. Mori: Antiangiogenic activity of nasunin, an antioxidant anthocyanin, in eggplant peels. J Agric Food Chem 53, 6272-6275 (2005)
doi:10.1021/jf050796r
PMid:16076105
13. K. Matsubara, M. Mori and Y. Mizushina: Petasiphenol which inhibits DNA polymerase lambda activity is an inhibitor of in vitro angiogenesis. Oncol Rep 11, 447-451 (2004)
PMid:14719082
14. K. Matsubara, H. Matsumoto, Y. Mizushina, M. Mori, N. Nakajima, M. Fuchigami, H. Yoshida and T. Hada: Inhibitory effect of glycolipids from spinach on in vitro and ex vivo angiogenesis. Oncol Rep 14, 157-160 (2005)
PMid:15944783
15. K. Matsubara, T. Kayashima, M. Mori, H. Yoshida and Y. Mizushina: Inhibitory effects of vitamin K3 on DNA polymerase and angiogenesis. Int J Mol Med 22, 381-387 (2008)
PMid:18698499
16. T. Kayashima, M. Mori, H. Yoshida, Y. Mizushina and K. Matsubara: 1,4-Naphthoquinone is a potent inhibitor of human cancer cell growth and angiogenesis. Cancer Lett 278, 34-40 (2009)
doi:10.1016/j.canlet.2008.12.020
PMid:19168278
17. T. Kayashima, M. Mori, R. Mizutani, K. Nishio, K. Kuramochi, K. Tsubaki, H. Yoshida, Y. Mizushina and K. Matsubara: Synthesis and biological evaluation of vitamin K derivatives as angiogenesis inhibitor. Bioorg Med Chem 18, 6305-6309 (2010)
doi:10.1016/j.bmc.2010.07.022
18. A. Kornberg and T. A. Baker: DNA replication, second edition, W. H. Freeman (ed), New York, Chapter 6, 197-225 (1992)
19. K. Bebenek and T. A. Kunkel: DNA Repair and Replication: Advances in Protein Chemistry, W. Yang (ed), San Diego, 137-165 (2004)
doi:10.1016/S0065-3233(04)69005-X
20. E. C. Friedberg, W. J. Feaver and V. L. Gerlach: The many faces of DNA polymerases: strategies for mutagenesis and for mutational avoidance. Proc Natl Acad Sci USA 97, 5681-5683 (2000)
doi:10.1073/pnas.120152397
PMid:10811923
21. A. G. So and K. M. Downey: Eukaryotic DNA replication. Crit Rev Biochem Mol Biol 27, 129-155 (1992)
doi:10.3109/10409239209082561
PMid:1727692
22. K. Sakaguchi, F. Sugawara and Y. Mizushina: Inhibitors of eukaryotic DNA polymerases. Seikagaku 74, 244-251 (2002)
PMid:11974919
23. G. Maga and U. Hubscher: Repair and translesion DNA polymerases as anticancer drug targets. Anticancer Agents Med Chem 8, 431-447 (2008)
PMid:18473728
24. Y. Mizushina, A. Saito, A. Tanaka, N. Nakajima, I. Kuriyama, M. Takemura, T. Takeuchi, F. Sugawara and H. Yoshida: Structural analysis of catechin derivatives as mammalian DNA polymerase inhibitors. Biochem Biophys Res Commun 333, 101-109 (2005)
doi:10.1016/j.bbrc.2005.05.093
PMid:15950188
25. K. Matsubara, A. Saito, A. Tanaka, N. Nakajima, R. Akagi, M. Mori and Y. Mizushina: Catechin conjugated with fatty acid inhibits DNA polymerase and angiogenesis. DNA Cell Biol 25, 95-103 (2006)
doi:10.1089/dna.2006.25.95
PMid:16460233
26. K. Matsubara, A. Saito, A. Tanaka, N. Nakajima, R. Akagi, M. Mori and Y. Mizushina: Epicatechin conjugated with fatty acid is a potent inhibitor of DNA polymerase and angiogenesis. Life Sci 80, 1578-1585 (2007)
doi:10.1016/j.lfs.2007.01.049
PMid:17328920
27. K. Tamai, K. Kojima, T. Hanaichi, S. Masaki, M. Suzuki, H. Umekawa and S. Yoshida: Structural study of immunoaffinity-purified DNA polymerase alpha-DNA primase complex from calf thymus. Biochim Biophys Acta 950, 263-273 (1988)
PMid:3167053
28. T. Date, M. Yamaguchi, F. Hirose, Y. Nishimoto, K. Tanihara and A. Matsukage: Expression of active rat DNA polymerase beta in Escherichia coli. Biochemistry 27, 2983-2990 (1988)
doi:10.1021/bi00408a048
PMid:3042024
29. S. Umeda, T. Muta, T. Ohsato, C. Takamatsu, N. Hamasaki and D. Kang: The D-loop structure of human mtDNA is destabilized directly by 1-methyl-4-phenylpyridinium ion (MPP+), a parkinsonism-causing toxin. Eur J Biochem 267, 200-206 (2000)
doi:10.1046/j.1432-1327.2000.00990.x
PMid:10601867
30. M. Oshige, R. Takeuchi, R. Ruike, K. Kuroda and K. Sakaguchi: Subunit protein-affinity isolation of Drosophila DNA polymerase catalytic subunit: Protein Expr Purif 35, 248-256 (2004)
doi:10.1016/j.pep.2004.02.001
PMid:15135399
31. N. Aoyagi, S. Matsuoka, A. Furunobu, A. Matsukage and K. Sakaguchi: Drosophila DNA polymerase delta: Purification and characterization. J Biol Chem 269, 6045-6050 (1994)
PMid:7907087
32. N. Aoyagi, M. Oshige, F. Hirose, K. Kuroda, A. Matsukage and K. Sakaguchi: DNA polymerase epsilon from Drosophila melanogaster. Biochem Biophys Res Commun 230, 297-301 (1997)
doi:10.1006/bbrc.1996.5945
PMid:9016770
33. R.K. Hamatake, H. Hasegawa, A.B. Clark, K. Bebenek, T.A. Kunkel and A. Sugino: Purification and characterization of DNA polymerase II from the yeast Saccharomyces cerevisiae. Identification of the catalytic core and a possible holoenzyme form of the enzyme. J Biol Chem 265, 4072-4083 (1990)
PMid:2406268
34. K. Sakaguchi, Y. Hotta and H. Stern: Chromatin-associated DNA polymerase activity in meiotic cells of lily and mouse. Cell Struct Funct 5, 323-334 (1980)
doi:10.1247/csf.5.323
35. Y. Mizushina, H. Yagi, N. Tanaka, T. Kurosawa, H. Seto, K. Katsumi, M. Onoue, H. Ishida, A. Iseki, T. Nara, K. Morohashi, T. Horie, Y. Onomura, M. Narusawa, N. Aoyagi, K. Takami, M. Yamaoka, Y. Inoue, A. Matsukage, S. Yoshida and K. Sakaguchi: Screening of inhibitor of eukaryotic DNA polymerases produced by microorganisms. J Antibiot (Tokyo) 49, 491-492 (1996)
36. Y. Mizushina, N. Tanaka, H. Yagi, T. Kurosawa, M. Onoue, H. Seto, T. Horie, N. Aoyagi, M. Yamaoka, A. Matsukage, S. Yoshida and K. Sakaguchi: Fatty acids selectively inhibit eukaryotic DNA polymerase activities in vitro. Biochim Biophys Acta 1308, 256-262 (1996)
PMid:8809118
37. Y. Mizushina, S. Yoshida, A. Matsukage and K. Sakaguchi: The inhibitory action of fatty acids on DNA polymerase beta. Biochim Biophys Acta 1336, 509-521 (1997)
PMid:9367179
38. A. Ogawa, T. Murate, M. Suzuki, Y. Nimura and S. Yoshida: Lithocholic acid, a putative tumor promoter, inhibits mammalian DNA polymerase beta. Jpn J Cancer Res 89, 1154-1159 (1998)
PMid:9914784
39. K. Tamiya-Koizumi, T. Murate, M. Suzuki, C. G. Simbulan, M. Nakagawa, M. Takemura, K. Furuta, S. Izuta and S. Yoshida: Inhibition of DNA primase by sphingosine and its analogues parallels with their growth suppression of cultured human leukemic cells. Biochem Mol Biol Int 41, 1179-1189 (1997)
PMid:9161713
40. C. Nakayama and M. Saneyoshi: Inhibitory effects of 9-β-D-xylofuranosyladenine 5'-triphosphate on DNA-dependent RNA polymerase I and II from cherry salmon (Oncorhynchus masou). J Biochem (Tokyo) 97, 1385-1389 (1985)
PMid:2411720
41. D. A. Soltis and O. C. Uhlenbeck: Isolation and characterization of two mutant forms of T4 polynucleotide kinase. J Biol Chem 257, 11332-11339 (1982)
PMid:6288679
42. B. C. Lu and K. Sakaguchi: An endo-exonuclease from meiotic tissues of the basidiomycete Coprinus cinereus: Its purification and characterization. J Biol Chem 266, 21060-21066 (1991)
PMid:1657947
43. T. Mosmann: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 55-63 (1983)
doi:10.1016/0022-1759(83)90303-4
44. M. Mori, Y. Sadahira, S. Kawasaki, T. Hayashi, K. Notohara and M. Awai: Capillary growth from reversed rat aortic segments cultured in collagen gel. Acta Pathol Jpn 38, 1503-1512 (1988)
PMid:2467510
45. S. Kawasaki, M. Mori and M. Awai: Capillary growth of rat aortic segments cultured in collagen gel without serum. Acta Pathol Jpn 39, 712-718 (1989)
PMid:2482651
46. Y. Cao and R. Cao: Angiogenesis inhibited by drinking tea. Nature 398, 381 (1999)
doi:10.1038/18793
PMid:10201368
47. R. F. Nicosia and A. Ottinetti: Growth of microvessels in serum-free matrix culture of rat aorta. Lab Invest 63, 115-122 (1990)
PMid:1695694
48. E. A. Kruger, P. H. Duray, M. G. Tsokos, D. J. Venzon, S. K. Libutti, S. C. Dixon, M. A. Rudek, J. Pluda, C. Allerga and W. D. Figg: Endostatin inhibits microvessel formation in the ex vivo rat aortic ring angiogenesis assay. Biochem Biophys Res Commun 268, 183-191 (2000)
doi:10.1006/bbrc.1999.2018
PMid:10652234
49. T. G. Zogakis, N. G. Costouros, E. A. Kruger, S. Forbes, M. He, M. Qian, A. L. Feldman, W. D. Figg, H. R. Alexander, E. T. Liu, E. C. Kohn and S. K. Libutti: Microarray gene expression profiling of angiogenesis inhibitors using the rat aortic ring assay. Biotechniques 33, 664-666, 668, 670 (2002)
PMid:12238776
50. Y. Mizushina: Specific inhibitors of mammalian DNA polymerase species. Biosci Biotechnol Biochem 73, 1239-1251 (2009)
doi:10.1271/bbb.90121
51. A. H. Jr. Vagnucci and W. W. Li: Alzheimer's disease and angiogenesis. Lancet 361, 605-608 (2003)
doi:10.1016/S0140-6736(03)12521-4
52. Y. Cao, R. Cao and E. Bråkenhielm: Antiangiogenic mechanisms of dietderived polyphenols. J Nutr Biochem 13, 380-390 (2002)
doi:10.1016/S0955-2863(02)00204-8
53. S. Uesato, Y. Kitagawa, Y. Hara, H. Tokuda, M. Okuda, Y. X. Mou, T. Mukainaka and H. Nishino: Antitumor promoting activities of 3-Oacyl-(−) epigallocatechins. Bioorg Med Chem Lett 10, 1673-1675 (2000)
doi:10.1016/S0960-894X(00)00314-0
54. J. P. E. Spencer, H. Schroeter, G. Kuhnle, S. K. S. Srai, R. M. Tyrrell, U. Hahn and C. Rice-Evans: Epicatechin and its in vivo metabolite, 3'-O-methyl epicatechin, protect human fibroblasts from oxidative-stress-induced cell death involving caspase-3 activation. Biochem J 354, 493-500 (2001)
doi:10.1042/0264-6021:3540493
PMid:11237853 PMCid:1221680
55. S. Lamy, D. Gingras and R. Béliveau: Green tea catechins inhibit vascular endothelial growth factor receptor phosphorylation. Cancer Res 62, 381-385 (2000)
56. K. Murakami-Murofushi, S. Kobayashi, K. Onimura, M. Matsumoto, M. Shioda, S. Yoshida, M. Shoji and H. Murofushi: Selective inhibition of DNA polymerase-alpha family with chemically synthesized derivatives of PHYLPA, a unique Physarum lysophosphatidic acid. Biochim Biophys Acta 1258, 57-60 (1995)
PMid:7654781
57. A. Patti, C. Sanfilippo, M. Piattelli and G. Nicolosi: Use of Mucor miehei lipase in the preparation of long chain 3-O-acylcatechins. J Mol Catal B: Enzym 10, 577-582 (2000)
doi:10.1016/S1381-1177(00)00140-5
Abbreviations: EGCG: Epigallocatechin-3-O-gallate, DMSO: dimethyl sulfoxide, dNTP: 2'-deoxynucleoside 5'-triphosphate, dsDNA: double-stranded DNA, dTTP: 2'-deoxythymidine 5'-triphosphate, HUVEC: human umbilical vein endothelial cell, pol: DNA polymerase, VEGF: vascular endothelial growth factor
Key Words: Acylated catechin derivative, Angiogenesis, Anti-cancer, Catechin, Cultured cell growth, DNA polymerase, E.C.2.7.7.7, Enzyme inhibitor, HUVEC
Send correspondence to: Yoshiyuki Mizushina, Laboratory of Food and Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, 518 Arise, Ikawadani-cho, Nishi-ku, Kobe, Hyogo 651-2180, Japan, Tel: 81-78-974-1551 (ext. 3232), Fax: 81-78-974-5689, E-mail:mizushin@nutr.kobegakuin.ac.jp